Cytoskeletal stiffness, friction, and fluidity of cancer cell lines with different metastatic potential.

We quantified mechanical properties of cancer cells differing in metastatic potential. These cells included normal and H-ras-transformed NIH3T3 fibroblast cells, normal and oncoprotein-overexpressing MCF10A breast cancer cells, and weakly and strongly metastatic cancer cell line pairs originating from human cancers of the skin (A375P and A375SM cells), kidney (SN12C and SN12PM6 cells), prostate (PC3M and PC3MLN4 cells), and bladder (253J and 253JB5 cells). Using magnetic twisting cytometry, cytoskeletal stiffness (g') and internal friction (g″) were measured over a wide frequency range. The dependencies of g' and g″ upon frequency were used to determine the power law exponent x which is a direct measure of cytoskeletal fluidity and quantifies where the cytoskeleton resides along the spectrum of solid-like (x = 1) to fluid-like (x = 2) states. Cytoskeletal fluidity x increased following transformation by H-ras oncogene expression in NIH3T3 cells, overexpression of ErbB2 and 14-3-3-ζ in MCF10A cells, and implantation and growth of PC3M and 253J cells in the prostate and bladder, respectively. Each of these perturbations that had previously been shown to enhance cancer cell motility and invasion are shown here to shift the cytoskeleton towards a more fluid-like state. In contrast, strongly metastatic A375SM and SN12PM6 cells that disseminate by lodging in the microcirculation of peripheral organs had smaller x than did their weakly metastatic cell line pairs A375P and SN12C, respectively. Thus, enhanced hematological dissemination was associated with decreased x and a shift towards a more solid-like cytoskeleton. Taken together, these results are consistent with the notion that adaptations known to enhance metastatic ability in cancer cell lines define a spectrum of fluid-like versus solid-like states, and the position of the cancer cell within this spectrum may be a determinant of cancer progression.

Low intensity ultrasound perturbs cytoskeleton dynamics.

Therapeutic ultrasound is widely employed in clinical applications but its mechanism of action remains unclear. Here we report prompt fluidization of a cell and dramatic acceleration of its remodeling dynamics when exposed to low intensity ultrasound. These physical changes are caused by very small strains (10-5) at ultrasonic frequencies (106 Hz), but are closely analogous to those caused by relatively large strains (10-1) at physiological frequencies (100 Hz). Moreover, these changes are reminiscent of rejuvenation and aging phenomena that are well-established in certain soft inert materials. As such, we suggest cytoskeletal fluidization together with resulting acceleration of cytoskeletal remodeling events as a mechanism contributing to the salutary effects of low intensity therapeutic ultrasound.

Dynamics of the cytoskeleton: how much does water matter?

The principal constituent of the living cell is water. The role of the hydration shell and bulk H(2)O solvent is well recognized in the dynamics of isolated proteins, but the role of water in the dynamics of the integrated living cytoskeleton (CSK) remains obscure. Here we report a direct connection of dynamics of water to dynamics of the integrated CSK. The latter are known to be scale-free and to hinge upon a frequency f(0) that is roughly invariant across cell types. Although f(0) is comparable in magnitude to the rotational relaxation frequency of water (gigahertz range), the physical basis of f(0) remains unknown. Using the human airway smooth muscle cell as a model system, we show here that replacing water acutely with deuterium oxide impacts CSK dynamics in major ways, slowing CSK remodeling dynamics appreciably, and lowering f(0) by up to four orders of magnitude. Although these observations do not distinguish contributions of bulk solvent versus hydration shell, they suggest a unifying hypothesis, namely, that dynamics of integrated CSK networks are slaved in a direct fashion to fluctuations arising in intracellular water.

Stretch magnitude and frequency-dependent actin cytoskeleton remodeling in alveolar epithelia.

Alveolar epithelial cells (AEC) maintain integrity of the blood-gas barrier with gasket-like intercellular tight junctions (TJ) that are anchored internally to the actin cytoskeleton. We hypothesize that stretch rapidly reorganizes actin (<10 min) into a perijunctional actin ring (PJAR) in a manner that is dependent on magnitude and frequency of the stretch, accompanied by spontaneous movement of actin-anchored receptors at the plasma membrane. Primary AEC monolayers were stretched biaxially to create a change in surface area (DeltaSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min, or held tonic at 25% DeltaSA for up to 60 min, or left unstretched. By 10 min of stretch PJARs were evident in 25% and 37% DeltaSA at 0.25 Hz, but not for 12% DeltaSA at 0.25 Hz, or at tonic 25% DeltaSA, or with no stretch. Treatment with 1 muM jasplakinolide abolished stretch-induced PJAR formation, however. As a rough index of remodeling rate, we measured spontaneous motions of 5-mum microbeads bound to actin focal adhesion complexes on the apical membrane surfaces; within 1 min of exposure to DeltaSA of 25% and 37%, these motions increased substantially, increased with increasing stretch frequency, and were consistent with our mechanistic hypothesis. With a tonic stretch, however, the spontaneous motion of microbeads attenuated back to unstretched levels, whereas PJAR remained unchanged. Stretch did not increase spontaneous microbead motion in human alveolar epithelial adenocarcinoma A549 monolayers, confirming that this actin remodeling response to stretch was a cell-type specific response. In summary, stretch of primary rat AEC monolayers forms PJARs and rapidly reorganized actin binding sites at the plasma membrane in a manner dependent on stretch magnitude and frequency.

Mechanosensing of substrate thickness.

Structure and function of the adherent cell depend in a crucial way on its microenvironment, including the stiffness of its substrate. It is often asserted that substrate thickness (as opposed to stiffness) plays a negligible role and therefore may be considered semi-infinite. This assertion has been recently challenged, but the characteristic length scale to consider in this regard is poorly understood. We show here that this characteristic length scale is the lateral cell size. As substrate thickness approaches the lateral dimension of the cell, the apparent stiffness of the substrate is amplified to levels much greater than the intrinsic stiffness of the substrate. This change in apparent stiffness is readily sensed by the cell, leading to changes in cell spreading area, stiffness, and contractile forces. In contrast to these responses that occur over the length of the cell, mechanosensing around an isolated point force is influenced greatly by intrinsic substrate stiffness but to a negligible extent by substrate thickness. We conclude that mechanosensing of substrate thickness is dominated in large part by traction forces spread over the lateral dimension of the cell.

Remodeling of integrated contractile tissues and its dependence on strain-rate amplitude.

Here we investigate the origin of relaxation times governing the mechanical response of an integrated contractile tissue to imposed cyclic changes of length. When strain-rate amplitude is held constant as frequency is varied, fast events are accounted for by actomyosin cross-bridge cycling, but slow events reveal relaxation processes associated with ongoing cytoskeletal length adaptation. Although both relaxation regimes are innately nonlinear, these regimes are unified and their positions along the frequency axis are set by the imposed strain-rate amplitude.

Reinforcement versus fluidization in cytoskeletal mechanoresponsiveness.

Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment.

Modulation of host cell mechanics by Trypanosoma cruzi.

To investigate the effects of Trypanosoma cruzi on the mechanical properties of infected host cells, cytoskeletal stiffness and remodeling dynamics were measured in parasite-infected fibroblasts. We find that cell stiffness decreases in a time-dependent fashion in T. cruzi-infected human foreskin fibroblasts without a significant change in the dynamics of cytoskeletal remodeling. In contrast, cells exposed to T. cruzi secreted/released components become significantly stiffer within 2 h of exposure and exhibit increased remodeling dynamics. These findings represent the first direct mechanical data to suggest a physical picture in which an intact, stiff, and rapidly remodeling cytoskeleton facilitates early stages of T. cruzi invasion and parasite retention, followed by subsequent softening and disassembly of the cytoskeleton to accommodate intracellular replication of parasites. We further suggest that these changes occur through protein kinase A and inhibition of the Rho/Rho kinase signaling pathway. In the context of tissue infection, changes in host cell mechanics could adversely affect the function of the infected organs, and may play an important role on the pathophysiology of Chagas' disease.

Airway smooth muscle and bronchospasm: fluctuating, fluidizing, freezing.

We review here four recent findings that have altered in a fundamental way our understanding of airways smooth muscle (ASM), its dynamic responses to physiological loading, and their dominant mechanical role in bronchospasm. These findings highlight ASM remodeling processes that are innately out-of-equilibrium and dynamic, and bring to the forefront a striking intersection between topics in condensed matter physics and ASM cytoskeletal biology. By doing so, they place in a new light the role of enhanced ASM mass in airway hyper-responsiveness as well as in the failure of a deep inspiration to relax the asthmatic airway. These findings have established that (i) ASM length is equilibrated dynamically, not statically; (ii) ASM dynamics closely resemble physical features exhibited by so-called soft glassy materials; (iii) static force-length relationships fail to describe dynamically contracted ASM states; (iv) stretch fluidizes the ASM cytoskeleton. Taken together, these observations suggest that at the origin of the bronchodilatory effect of a deep inspiration, and its failure in asthma, may lie glassy dynamics of the ASM cell.

Out-of-equilibrium dynamics in the cytoskeleton of the living cell.

We report here measurements of rheological properties of the human airway smooth muscle cell using forced nanoscale motions of Arg-Gly-Asp RGD-coated microbeads tightly bound to the cytoskeleton. With changes of forcing amplitude, the storage modulus showed small but systematic nonlinearities, especially after treatment with a contractile agonist. In a dose-dependent manner, a large oscillatory shear applied from a few seconds up to 400 s caused the cytoskeleton matrix to soften, a behavior comparable to physical rejuvenation observed in certain inert soft materials; the stiffness remained constant for as long as the large oscillatory shear was maintained, but suddenly fell with shear cessation. Stiffness then followed a slow scale-free recovery, a phenomenon comparable to physical aging. However, acetylated low-density lipoprotein acLDL-coated microbeads, which connect mainly to scavenger receptors, did not show similar out-of-equilibrium behaviors. Taken together, these data demonstrate in the cytoskeleton of the living cell behaviors with all the same signatures as that of soft inert condensed systems. This unexpected intersection of condensed matter physics and cytoskeletal biology suggests that trapping, intermittency, and approach to kinetic arrest represent central mesoscale features linking underlying molecular events to integrative cellular functions.

Directional memory and caged dynamics in cytoskeletal remodelling.

We report directional memory of spontaneous nanoscale displacements of an individual bead firmly anchored to the cytoskeleton of a living cell. A novel method of analysis shows that for shorter time intervals cytoskeletal displacements are antipersistent and thus provides direct evidence in a living cell of molecular trapping and caged dynamics. At longer time intervals displacements are persistent. The transition from antipersistence to persistence is indicative of a time-scale for cage rearrangements and is found to depend upon energy release due to ATP hydrolysis and proximity to a glass transition. Anomalous diffusion is known to imply memory, but we show here that memory is attributed to direction rather than step size. As such, these data are the first to provide a molecular-scale physical picture describing the cytoskeletal remodelling process and its rate of progression.

Cytoskeleton dynamics: fluctuations within the network.

Out-of-equilibrium systems, such as the dynamics of a living cytoskeleton (CSK), are inherently noisy with fluctuations arising from the stochastic nature of the underlying biochemical and molecular events. Recently, such fluctuations within the cell were characterized by observing spontaneous nano-scale motions of an RGD-coated microbead bound to the cell surface [Bursac et al., Nat. Mater. 4 (2005) 557-561]. While these reported anomalous bead motions represent a molecular level reorganization (remodeling) of microstructures in contact with the bead, a precise nature of these cytoskeletal constituents and forces that drive their remodeling dynamics are largely unclear. Here, we focused upon spontaneous motions of an RGD-coated bead and, in particular, assessed to what extent these motions are attributable to (i) bulk cell movement (cell crawling), (ii) dynamics of focal adhesions, (iii) dynamics of lipid membrane, and/or (iv) dynamics of the underlying actin CSK driven by myosin motors.

Deformability, dynamics, and remodeling of cytoskeleton of the adherent living cell.

A trail of evidence has led to an unexpected intersection of topical issues in condensed matter physics and cytoskeletal biology. On the one hand, the glass transition and the jammed state are two outstanding unsolved problems; such systems are out-of-equilibrium, disordered, and their transitions between solid-like and liquid-like states are not understood. On the other hand, cellular systems are increasingly being considered as interconnected maps of protein interactions that are highly specific and tightly regulated but, even when such comprehensive maps become available, they may be insufficient to define biological function at the integrative level because they do not encompass principles that govern dynamics at intermediate (meso) scales of organization. It is interesting, therefore, that the cytoskeleton of the living cell shows physical properties and remodeling dynamics with all the same signatures as soft inert condensed systems, although with important differences as well. Data reviewed here suggest that trapping, intermittency, and approach to kinetic arrest represent mesoscale features of collective protein-protein interactions linking underlying molecular events to integrative cellular functions such as crawling, contraction and remodeling. Because these are crucial cell functions, this synthesis may offer new perspectives on a variety of disorders including infectious disease, cardiovascular disease, asthma and cancer.

Cytoskeletal remodelling and slow dynamics in the living cell.

The cytoskeleton (CSK) is a crowded network of structural proteins that stabilizes cell shape and drives cell motions. Recent studies on the dynamics of the CSK have established that a wide variety of cell types exhibit rheology in which responses are not tied to any particular relaxation times and are thus scale-free. Scale-free rheology is often found in a class of materials called soft glasses, but not all materials expressing scale-free rheology are glassy (see plastics, wood, concrete or some metals for example). As such, the extent to which dynamics of the CSK might be regarded as glassy remained an open question. Here we report both forced and spontaneous motions of microbeads tightly bound to the CSK of human muscle cells. Large oscillatory shear fluidized the CSK matrix, which was followed by slow scale-free recovery of rheological properties (aging). Spontaneous bead motions were subdiffusive at short times but superdiffusive at longer times; intermittent motions reflecting nanoscale CSK rearrangements depended on both the approach to kinetic arrest and energy release due to ATP hydrolysis. Aging, intermittency, and approach to kinetic arrest establish a striking analogy between the behaviour of the living CSK and that of inert non-equilibrium systems, including soft glasses, but with important differences that are highly ATP-dependent. These mesoscale dynamics link integrative CSK functions to underlying molecular events, and represent an important intersection of topical issues in condensed matter physics and systems biology.